Protein phosphorylation is a critical post-translational modification that cells use to regulate many biological processes, including signaling, growth and division. Consistent with its role in cellular function, aberrant protein phosphorylation has been implicated in many diseases including cancer and heart disease. However, efficient capture and analysis of proteome-wide phosphorylation patterns remains an unsolved problem. In this NRSA postdoctoral fellowship (F32) proposal, we aim to solve the challenges of performing a proteome-wide analysis of protein phosphorylation by developing a class of multivalent nanoparticle materials capable of selectively enriching phosphoproteins out of a complex biological sample followed by top-down proteomic analysis of the enriched intact proteins. Our approach combines recent advances in both nanoparticle chemistry and mass-spectrometry-based proteomics and challenges or improves on existing conventional methods by offering an inexpensive platform which exhibits: 1) high capturing capacity, efficiency, and selectivity, 2) universal binding of all phosphoproteins, 3) scalability and reusability, and 4) the ability to preserve native activity following enrichment. For the enrichment we will synthesize metal ferrite magnetic nanoparticles with diameters <10 nm functionalized with phosphate-specific binding groups which can selectively and reversibly bind phosphoproteins. Following capture and enrichment, intact proteins will be released and purified by liquid chromatography and analyzed by high-resolution mass spectrometry. The chemistry of protein enrichment will be optimized by systematically studying the surface chemistry of the particles and binding/elution buffer conditions on a control mixture of (phospho)proteins. To investigate the capabilities of the platform on a biological sample, we will demonstrate its use on the analysis of the human heart proteome where filament protein phosphorylation is critical to muscle function. The success of this proposal will not only provide a diverse and multidisciplinary research experience, it will also provide a powerful and easy to use technology for both the research and medical communities for the analysis of protein phosphorylation in biological samples. PUBLIC HEALTH RELEVANCE: This proposal seeks to develop a new multivalent nanomaterial for the selective and efficient capture of phosphoproteins from complex biological mixtures followed by comprehensive proteomic analysis of enriched intact phosphoproteins. The success of this work will have significant impacts in the medical and clinical diagnostic communities where protein phosphorylation may serve as a biomarker for a specific condition or disease, and will provide a valuable tool for understanding the molecular chemistry of many diseases by allowing the characterization of phosphorylation-based cell signaling pathways.